Agrobacterium is a gram negative soil bacterium that transfers its Ti plasmid or the ‘Tumor inducing plasmid’ into the cells of most dicotyledonous plants and quite a good number of monocotyledonous plants. The Ti plasmid undergoes cell-cell recognition, signal transduction, cellular and nuclear import and finally T-DNA integration (Winans S C Two-way chemical signaling in Agrobacterium-plant interactions Microbiological-Reviews. 1992, 56: 1, 12-31).
The transfer DNA or the T-DNA harbouring the oncogenes bounded by the 25 bp imperfect border repeats on either side brings about genetic transformation and hence crown gall disease. The process of signal transduction is initiated by a set of several ‘virulence genes’, of which 7 genes are the most important.
The first step of signal reception and then transduction is triggered by the inducers like phenolics and sugars (Ankenbauer R G; Nester E W Sugar-mediated induction of Agrobacterium tumefaciens virulence genes: structural specificity and activities of monosaccharides. Journal of Bacteriology. 1990, 172: 11, 6442-6446).
For the past two decades, revolutionary work has been done on development of genetically transformed plants wherein, the disarmed strains of Agrobacterium (i. e. the Ti plasmid with the oncogenes replaced by genes of interest) are employed to produce plants as per ones requirements at a much shorter time. The inducers that are used for such genetic transformation experiments are generally the Acetosyringone (Morris J W; Morris R O Identification of an Agrobacterium tumefaciens virulence gene inducer from the Pinaceous gymnosperm Pseudotsuga menziesii. Proceedings of the National Academy of Sciences of the United States of America 1990, 87: 9, 3614-3618) and the Hydroxy-acetosyringone that are commercially provided by the Sigma Aldrich Company, USA.
Tea leaves when extracted, are known to have fractions of caffeine, catechins and other flavonols and amino acids. Some chance experiments indicated that particular fractions of tea containing caffeine are capable of promoting infection by Agrobacterium, cell-cell recognition and virulence. This led us to believe that the caffeine fractions can be used as a virulence inducer during genetic transformation experiments instead of Acetosyringone or Hydroxy-acetosyringone.
Besides containing high levels of the 6 types of catechins (C) and their derivatives viz., epicatechin (EC), gallocatechins (GC), epigallocatechins (EGC), epicatechin gallate (ECg), epigallocatechin gallate (EGCg), tea leaves also contain caffeine, amino acids, nitrogenous compounds, vitamins, inorganic elements, carbohydrates and lipids (Chu D C and Juneja L. R. General chemical composition of green tea and its infusion. In: Chemistry and Application of Green Tea. 1997. CRC Press, N. York. eds. Yamamoto T., Juneja L. R., Chu D C, Kim M., pp.).
The report of Sunilkumar et al., 1999. (Sunilkumar G; Vijayachandra K; Veluthambi K 1999, Pre-incubation of cut tobacco leaf explants promotes Agrobacterium-mediated transformation by increasing vir gene induction. Plant Science Limerick. 141: 1, 51-58) indicated that the requirement of preincubation for increased transformation efficiency can be obviated by the addition of 100 μM acetosyringone to the freshly cut leaf rings during the co-cultivation with Agrobacterium. The production of vir gene inducers by the leaf rings during the pre-incubation period is an important factor that contributes to increased transformation efficiency of Agrobacterium upon pre-incubation but the drawback is that the effect of the inducer ‘acetosyringone’ and pre-incubation is similar and thus inducers do not have much role to play.
Expression of Agrobacterium tumefaciens virulence (vir) genes and transformation of dicots by this organism are dependent upon host plant phenolic compounds and several alkylsyringamides, syringic acid, synthetic amides like ethylsyringamide, ferulic or sinapic acids are powerful inducers of vir genes (Vir gene inducing activities of hydroxycinnamic acid amides in Agrobacterium tumefaciens Berthelot, K; Buret, D; Guerin, B; Delay, D; Negrel, J; Delmotte, F M. Phytochemistry. 1998, 49: 6, 1537-1548).
However, none of the inducers tested exhibited higher activity than acetosyringone, the reference compound for vir gene induction, with the exception to ethylsyringamide at concentrations above 1 mM. When tested on A. tumefaciens strain A348 (pSM243cd), ethylferulamide and ethylsinapamide are more efficient than the corresponding phenolic acids but only above 100 μM.
The major draw back is that the above mentioned inducers are very expensive chemical compounds that are required to be used at high concentrations above 100 μM. Moreover, the compounds like acetosyringone are manufactured only by a few select companies like Sigma Aldrich and requires import from United States of America by research laboratories situated in developing countries.
Lee et al., 1995 (Lee, YongWoog; Jin, ShouGuang; Sim, WoongSeop; Nester, E W; Lee, Y W; Jin, S G; Sim, W S Proceedings of the National Academy of Sciences of the United States of America. 1995 Genetic evidence for direct sensing of phenolic compounds by the VirA protein of Agrobacterium tumefaciens. 92: 26, 12245-12249.) reported that the virulence (vir) genes of Agrobacterium tumefaciens are induced by low-molecular-weight phenolic compounds and monosaccharides through a two-component regulatory system consisting of the VirA and VirG proteins. The vir-inducing abilities of 15 different phenolic compounds like acetovanillone was tested using four wild-type strains of A. tumefaciens KU12, C58, A6, and Bo542. By transferring different Ti plasmids into isogenic chromosomal backgrounds, the phenolic-sensing determinant was shown to be associated with Ti plasmid. Subcloning of Ti plasmid indicates that the vira locus determines which phenolic compounds can function as vir gene inducers. These results suggest that the VirA protein directly senses the phenolic compounds for vir gene activation. The drawback of this report is that subcloning of Ti plasmid is required for the identification of the accurate phenolics inducer for vir a locus.
Hess et al., 1991 (Hess, K M; Dudley, M W; Lynn, D G; Joerger, R D; Binns, A N. 1991, Mechanism of phenolic activation of Agrobacterium virulence genes: development of a specific inhibitor of bacterial sensor/response systems. Proceedings of the National Academy of Sciences of the United States of America. 1991, 88: 17, 7854-7858) reported that the aglycone of the dihydrodiconiferyl alcohol glycosides were potent inducers of virulence gene expression in A. tumefaciens. Using this model, a specific inhibitor of vir induction was developed. The drawback of this report is that this inhibitor did not affect the induction of other genes on the Ti plasmid but irreversibly blocks vir expression.
Fortin et al., 1992 (Fortin, C; Nester, E W; Dion, P. 1992, Growth inhibition and loss of virulence in cultures of Agrobacterium tumefaciens treated with acetosyringone. Journal of Bacteriology. 174: 17, 5676-5685) reported that acetosyringone, a phenolic inducer of the virulence (vir) genes of A. tumefaciens, inhibited the growth of the nopaline type strains T37 and C58 incubated under acidic conditions. Two other vir inducers, sinapinic acid and syringaldehyde, also inhibited growth and promoted accumulation of avirulent clones in cultures of strains C58F and T37. On the other hand, various acetosyringone analogues reported not to induce the vir genes did not act as growth inhibitors. Mutants of strain C58F lacked the capacity to induce a virB::lacZ fusion in the presence of acetosyringone. The drawback of this report is that while some inducers were promotive, others are inhibitory and are also strain specific.
Delmotte et al., 1991 (Delmotte F M; Delay D; Cizeau J; Guerin B; Leple J C 1991. Agrobacterium vir-inducing activities of glycosylated acetosyringone, acetovanillone, syringaldehyde and syringic acid derivatives. Phytochemistry., 30: 11, 3549-3552.) reported that when A. tumefaciens str. A348 (pSM358) harbouring a virE::lacZ fusion plasmid was used to detect the ability of 13 synthetic acetosyringone, acetovanillone, syringaldehyde and syringic acid beta-glycosides to induce virulence, the activity of the reporter beta-galactosidase was detected by spectrofluorimetry using 4-methylumbelliferyl beta-galactopyranoside as substrate. Acetosyringonyl beta-L-fucopyranoside was the most active monoglycoside tested; even at high concentrations this compound was devoid of toxic effects. However, monoglycosides were less active vir inducers than free acetosyringone. In contrast, the beta-maltoside of syringaldehyde showed higher activity than the free phenol at high concentrations. The activity of such glycosylated inducers may be related to specific sugar receptors on the bacterial cell surface. The drawback of the report is that the acetosyringone is costly compound that need to be imported from Sigma Aldrich, USA.